Promotion Of Trophoblast Biological Functions By IL-35 Through Autophagy Under Hypoxic Starvation Conditions

Objective:In early gestation(<12 weeks of gestation),there is no significant blood flow in the placenta,resulting in a lack of oxygen and nutrient supply to the embryo.Thus,early placenta formation occurs in a physiologically hypoxic,nutrient-deficient environment.Therefore,as an important component of the placenta,extravillous trophoblast(EVT)needs to actively expand,invade the decidual tissue of the uterus,destroy its smooth muscle layer,remodel the maternal spiral arteries,form a vascular network,and create a low-resistance and high-flow uteroplacental circulation pattern.It can effectively exchange gas and nutrients,and create the best growth and development conditions for the embryo.However,for trophoblasts to function in early pregnancy under such harsh survival conditions,mechanisms to adapt to such stress are required.Autophagy,a cell survival strategy that allows cells to overcome various stressors such as starvation,hypoxia and viral infection,is a key intracellular catabolic process that provides energy for their own survival.Thus,trophoblasts avoid apoptosis by enhancing autophagy in a hostile environment as a way for trophoblasts to cope with the stress of survival.Interleukin-35(IL-35)is a recently identified cytokine in the IL-12 family,consisting of the IL12 subunit alpha chain(P35)and the IL-27 subunit Epstein-Barr virus-induced gene 3(EBI3)beta chain.Our previous study found that IL-35 is constitutively highly expressed in human early trophoblast cells and that high levels of IL-35 in peripheral blood in early pregnancy induce the conversion of B cells into IL-35+Breg cells,which are involved in the regulation of peripheral immune tolerance during pregnancy and are key players in the maintenance of normal pregnancy.But what is the effect of trophoblast-derived IL-35 on its own biological function?This has not been reported in the literature.Does autophagy promote embryonic growth and development in an environment of hypoxic starvation in early gestation?What is the role of IL-35 in the biological behaviour of trophoblast cells?Further studies are needed to confirm this.To address these scientific questions,this study investigates the mechanisms and signaling pathways involved in the effects of IL-35 on the biological functions of trophoblast cells through the promotion of autophagy under hypoxic starvation conditions,providing new targets and ideas for the early treatment and prevention of adverse pregnancy outcomes.Methods:1.The expression levels of IL-35 and its receptors gp130 and IL-12Rβ2 in trophoblast cells under normal culture conditions(5%CO2,37℃,20%O2)versus hypoxic starvation conditions(5%CO2,37℃,2%O2)were measured using qRT-PCR.2.The effect of IL-35 on the proliferation,migration and invasion of trophoblast cells under hypoxic starvation conditions was examined using the EDU,CCK8 and Transwell assays.3.Effect of IL-35 on trophoblast cell cycle and apoptosis under hypoxic starvation conditions using flow cytometry.4.The supernatants of trophoblast cells after exogenous IL-35 intervention under hypoxic starvation conditions were collected and co-cultured with HUVEC cells to observe the effect of IL-35 on the angiogenic ability of HUVEC cells under hypoxic starvation conditions.5.Effect of IL-35 on autophagy in trophoblast cells under hypoxic starvation conditions using MDC staining and Western blotting.6.Screening the concentration of autophagy inhibitor(3-MA)acting on trophoblast cells using CCK8,Western blotting assay.7.Effect of IL-35 on trophoblast proliferation,cell cycle and apoptosis after blocking autophagy by 3-MA,detected by CCK8 and flow cytometry.8.The effect of IL-35 on the mTOR-PI3K-AKT signalling pathway in trophoblast cells under hypoxic starvation conditions was examined using Western blotting assay.Results:1.Four trophoblast cell lines HTR-8/SVneo,JAR,JEG3 and BEWO express the subunit P35 of IL-35 with EBI3.2.Expression of IL-35 receptor gp130 and IL-12Rβ2 was upregulated under hypoxic starvation conditions,but IL-35 expression levels did not change significantly,suggesting that IL-35 is more sensitive to the action of trophoblast cells under hypoxic starvation conditions.3.Under hypoxic starvation conditions,the proliferative capacity of trophoblast cells was significantly inhibited,whereas IL-35 effectively promoted trophoblast cell proliferation,reversed the cell cycle G1 phase block caused by hypoxic starvation,promoted cell entry into S phase,and inhibited apoptosis,while IL-35 up-regulated the expression of proliferative molecules CDK2,CyclinE1,and Bcl-2 in HTR-8/SVneo and JAR cells,Bcl-2,and downregulated the expression of the inhibitory molecules P27,Bax,and Cleaved caspase3.4.Under hypoxic starvation conditions,the ability of trophoblast cells to cross the matrigel is reduced and migration and invasion functions are inhibited,while IL-35 can reverse this effect.5.Under hypoxic starvation conditions,IL-35 significantly promoted HUVEC cell angiogenesis,enhanced their tube-forming ability,regulated cell-cell communication between trophoblasts and vascular endothelial cells,and promoted the formation of placental microvascular network.6.Under hypoxic starvation conditions,IL-35 could enhance the occurrence of autophagy in trophoblast cells.After inhibiting autophagy using 3-MA,the protective ability of IL-35 on trophoblast cells was weakened,cell proliferation was inhibited and the rate of apoptosis was significantly increased.7.Western blotting experiments demonstrated that IL-35 promotes trophoblast autophagy by down-regulating the phosphorylation levels of p-mTOR,PI3K and p-AKT proteins under hypoxic starvation conditions.Conclusions:1.This study demonstrates that IL-35 is highly expressed in trophoblast cell lines.Under the conditions of hypoxic starvation in early pregnancy,trophoblast cells can promote their own proliferation,migration and invasion capacity by means of IL-35 autocrine,and act on vascular endothelial cells to promote spiral artery neogenesis and attenuate the damage caused by hypoxic starvation environment to trophoblast cells,thus playing an important regulatory role in maintaining embryonic development and physiological pregnancy.2.IL-35 induces and enhances autophagy in trophoblast cells by inhibiting the mTORPI3K-AKT signaling pathway,thereby promoting cell proliferation and migratory invasive capacity,inhibiting apoptosis,and promoting embryonic growth and development in the hypoxic starvation environment of early pregnancy.